Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.
Kesan sitotoksik sebatian organostanum (IV) terhadap pelbagai sel kanser telah dikaji oleh para saintis di seluruh dunia.Dalam kajian ini,dua sebatian baru organostanum (IV) iaitu difenilstanum (IV) etilfenilditiokarbamat (DFEF) dan difenilstanum (IV) butilfenilditiokarbamat (DFBF) telah diuji kesan sitotoksiknya terhadap sel eritroleukemia, K562. Sel eritroleukemia, K562 merupakan sel sasaran manakala, sel hepar Chang dan sel fibroblas V79 pula digunakan untuk menilai kesan kedua-dua sebatian ini terhadap sel bukan kanser. Kesan sitotoksik sebatian DFEF dan DFBF diuji menggunakan ujian asai 3-(4,5-dimetiltiazol-2-il)-2, 5-difeniltetrazolium bromida (MTT) dengan masa pendedahan 24 jam, 48 jam dan 72 jam pada kepekatan sebatian yang berbeza. Pemerhatian terhadap perubahan morfologi juga dilakukan menggunakan nilai IC50 yang diperolehi pada masa pendedahan seperti ujian asai MTT. Ujian sitotoksisiti telah menunjukkan sebatian DFEF dan DFBF adalah sangat toksik terhadap sel K562 dengan nilai IC50 kurang daripada 10 μM untuk ketiga-tiga masa pendedahan.Indeks pemilihan juga membuktikan bahawa kedua-dua sebatian memberikan kesan sitotoksik secara memilih terhadap sel K562 pada masa 48 jam dan 72 jam, tetapi pada masa 24 jam, sebatian ini bertindak secara tidak memilih terhadap sel K562 dan sel bukan kanser. Perubahan morfologi yang diperhatikan adalah menyerupai ciri-ciri apoptosis seperti pengecutan sel dan pembentukan jasad apoptotik dan juga nekrosis seperti sel lisis. Kesimpulannya, sebatian difenilstanum (IV) alkilfenilditiokarbamat berpotensi untuk dibangunkan sebagai agen antileukemia tetapi mekanisma khusus tindakan sebatian ini terhadap sel K562 perlu dikaji pada masa akan datang untuk menjelaskan potensi sebatian ini sebagai dadah antikanser yang baru.
Dietary changes that occur in response to economic development are collectively known as the nutrition transition. More specifically, diets built around staple cereals and tubers give way to diets with more animal products and more added sugars and fats. Although the proportion of dietary protein stays constant, plant proteins are replaced by animal proteins but in ways that are dependent on regional cultural, religious, and ethical concerns. The protein transition, viewed here as a subset of the broader nutrition transition, illustrates how dietary patterns in low- and middle-income countries are shaped by societal as well as by economic forces. The complexity of food decisions justifies the need to integrate nutrition with the social sciences in the study of evolving food systems.
Kajian ini dijalankan untuk melihat variasi taburan 210Po dan 210Pb melalui profil menegak aktivitinya, serta nisbah aktiviti 210Po/210Pb di dalam teras sedimen yang diambil di beberapa stesen persampelan pesisir pantai perairan Sarawak. Didapati profil taburan radionuklid tersebut dalam teras sedimen adalah berubah-ubah mengikut lokasi persampelan dan telah dibuktikan melalui analisis ANOVA yang menunjukkan bahawa terdapat perbezaan bererti pada 95% aras keyakinan bagi aktiviti 210Po (p = 0.000), 210Pb (p = 0.035) dan 210Po/210Pb (p = 0.000) di semua lokasi kajian. Secara umumnya, aktiviti 210Po dan 210Pb yang diukur masing-masing dalam julat 337 ke 2460 Bq/kg, 11 ke 84 Bq/kg di SR 01; 224 ke 2008 Bq/kg, 6 ke 80 Bq/kg di SR 02; 119 ke 1595 Bq/kg, 6 ke 84 Bq/kg di SR 03; 241 ke 2294 Bq/kg, 5 ke 82 Bq/kg di SR 04 dan 175 ke 1340 Bq/kg, 4 ke 44 Bq/kg di SR 05. Merujuk kepada julat tersebut, didapati aktiviti 210Po adalah lebih tinggi daripada aktiviti 210Pb dengan purata nisbah 210Po/210Pb di semua stesen adalah melebihi satu, iaitu dalam julat 20 ke 35. Variasi profil taburan radionuklid tersebut dipercayai dipengaruhi oleh beberapa faktor sekitaran dan telah dibuktikan terdapat korelasi yang kuat di antara taburan radionuklid dengan komposisi sedimen jenis kelodak (210Po:r = 0.701 dan 210Pb: r = 0.648), kedalaman air (210Po: r = -0.647) dan jarak stesen dari daratan (210Po: r = 0.746 dan 210Pb: r = 0.975). Oleh itu, dapat disimpulkan bahawa faktor-faktor tersebut merupakan penyumbang utama ke atas perubahan yang berlaku kepada taburan 210Po dan 210Pb.
Tujuan utama kajian ini adalah untuk menghasilkan bukti kesahan dan kebolehpercayaan metrik pengukuran kebergunaan perisian kursus matematik secara psikometrik dan seterusnya membentuk rumus skor kebergunaan. Kertas ini membincangkan pembentukan rumus kebergunaan. Berdasarkan pandangan secara teori, satu model hipotesis kebergunaan dengan tiga faktor iaitu kebolehgunaan, kefungsian dan kecekapan dan 85 metrik dicadangkan. Melalui pengesahan pakar dan ujian kebolehpercayaan metrik kebergunaan dikurangkan kepada 64. Instrumen dengan 64 metrik ditadbir ke atas 620 guru matematik di lima zon di Malaysia. Mereka diminta melayari perisian kursus matematik (PKM) sambil melengkapkan instrumen. Analisis faktor penjelajahan dilakukan untuk mengesahkan konstruk kebergunaan manakala analisis faktor pengesahan dijalankan untuk membentuk model berstruktur dengan memadankan 34 metrik dengan model hipotesis. Ujian-t dan ujian ANOVA mendapati tiga pembolehubah iaitu, bidang pengkhususan guru, pengalaman menggunakan komputer dan ralat penilaian menyumbang kepada skor kebergunaan. Dapatan ini digunakan untuk membentuk rumus kebergunaan PKM.
Penanaman semula hutan paya bakau dipertimbangkan sebagai satu usaha terbaik bagi melindungi dan memelihara kawasan pesisir pantai. Dalam kajian ini sejumlah 180 anak benih Bruguiera cylindrica telah ditanam di kawasan pesisir pantai Pulau Carey. Dua set eksperimen daripada segi corak dan medium dilakukan dengan 3 plot replika bagi setiap rawatan dengan kaedah Blok Rawak Lengkap. Kadar keterushidupan anak bakau B. cylindrica dicatat setiap 2 minggu selama 20 minggu. Keputusan menunjukkan corak penanaman secara kelompok adalah lebih sesuai (20%). Anak bakau yang ditanam secara langsung di atas tanah menunjukkan kadar hidup yang lebih tinggi (30%). Namun ujian analisis ANOVA menunjukkan tiada perbezaan yang signifikan antara ketiga-tiga corak taburan penanaman dan penggunaan medium yang berbeza ini terhadap keterushidupan anak bakau B. cylindrica.
Data bagi Sapindaceae dalam suatu plot kekal 50 ha di Hutan Simpan Pasoh, Negeri Sembilan, Malaysia telah diperoleh dari Institut Penyelidikan Hutan Malaysia (FRIM), Kepong dan telah diguna untuk menilai pengeluaran primer dan taburan famili. Lima bancian telah dijalankan dalam tahun 1985-2005 dan analisis data telah dijalankan ke atas bancian terakhir. Walau bagaimanapun perbandingan telah dibuat antara kelima-lima bancian untuk menentukan kevariabelan. Daripada bancian tahun 2005, 10 genus dan 18 spesies Sapindaceae telah direkodkan di dalam plot. Sejumlah 13360 dirian pokok telah dikira dan spesies yang paling banyak ialah Xerospermum noronhianum (7678 dirian). Jumlah biojisim bagi famili telah dianggarkan sebanyak 7.25 t/ha dan biojisim atas tanah tertinggi telah disumbangkan oleh X. noronhianum, dengan anggaran sebanyak 2.78 t/ha. Spesies ini juga mempunyai nilai keluasan pangkal tertinggi dengan 17.68 m2/ha. Anggaran biojisim untuk 20 tahun telah berkurangan secara signifikan (ANOVA, p<0.05) antara empat bancian. Genus yang mempunyai dirian tertinggi ialah Xerospermum (57.47%). Bagi melihat corak taburan, Indeks Taburan Morisita (Id) telah digunakan dan mendapati semua spesies mempunyai corak taburan secara rawak. Sebanyak 2292 dirian telah didapati mati (278/tahun) dan 1246 dirian baru direkodkan (172/tahun) pada tahun 2005. Secara keseluruhannya pertambahan saiz diameter bagi tempoh 20 tahun adalah sangat kecil iaitu 0.03 cm/tahun.
Illumination is one of the important physical aspects that influences comfortability during learning session particularly
among visually impaired students. The purpose of this study was to determine changes in illumination level in classrooms
during learning session at Sekolah Menengah Pendidikan Khas (SMPK), Setapak. The second objective was to compare
the illumination level in the classrooms under three different lighting conditions: daylight only, with additional artificial
light and with removal of obstructions to daylight. Illumination levels in 17 classrooms was measured at one hour interval,
between 8 am to 1 pm for the first stage and 19 classrooms under three different lighting conditions from 11 am to 12 noon
for the second stage, using ILM1335 (ISO-TECH, Taiwan) digital luxmeter. Illumination level increased significantly from
8 am to 11 am (One-Way Repeated Measures ANOVA: F(2.14, 34.26)=76.49, p<0 .001) and was maximum at 1 pm. The
illumination level was highest for the condition of daylight with additional artificial light (One-Way Repeated Measures
ANOVA: F(2,34)=110.51, p<0.001) compared to other conditions. Illumination levels for daylight without obstruction
was significantly higher than daylight only (pairwise comparison: p=0.001). Classroom illumination level was lowest
in the early morning. However, classroom illumination can be increased either by removing the obstructions to daylight
or with additional artificial lighting.
Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.
The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 °C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.
Heat sterilization of dairy products can promote the formation of advanced glycation end products (AGEs), protein oxidation products (POPs) and α-dicarbonyl compounds, which have a significant influence on health due to the close association of these products with diabetes complications. In this study, eight oat phenolic acids were first analyzed for their inhibitory effect against AGEs formation. Due to their strong inhibitory effects and structural differences, caffeic acid (CA) and gallic acid (GA) were further selected to assess their anti-glycosylation mechanisms using spectroscopy, chromatography and molecular docking. CA/GA reduced the production of total AGEs and POPs in various bovine milk simulation models and protected whey proteins from structural modifications, oxidation, and cross-linking. Comparative analyses showed a structure-effect relationship between CA/GA and AGEs inhibition. Oat phenolic acids against AGEs and POPs might be related to the unique bonding of key amino acid residues in whey proteins, the inhibitory role of early fructosamine and the trapping of reactive α-dicarbonyl groups to form adducts. In conclusion, oat phenolic acids might present a promising dietary strategy to alleviate AGEs production and glycation of proteins in dairy products upon storage.
Proteins often interact with each other to form complexes and play functional roles in almost all cellular processes. The study of protein-protein interactions is therefore critical to understand protein function and biological pathways. Affinity Purification coupled with Mass Spectrometry (AP-MS) is an invaluable technique for identifying the interaction partners in protein complexes. In this approach, the protein of interest is fused to an affinity tag, followed by the expression and purification of the fusion protein. The affinity-purified sample is then analyzed by mass spectrometry to identify the interaction partners of the bait proteins. In this chapter, we detail the protocol for tandem affinity purification (TAP) based on the use of the FLAG (a fusion tag with peptide sequence DYKDDDDK) and hemagglutinin (HA) peptide epitopes. The immunoprecipitation using dual-affinity tags offers the advantage of increasing the specificity of the purification with lower nonspecific-background interactions.
Fat-soluble vitamins (FSVs) offer a range of beneficial properties as important nutrients in human nutrition. However, the high susceptibility to environmental conditions such as high temperature, light, and oxygen leads to the degradation of these compounds. This review highlights the different formulations underlying the encapsulation of FSVs in biopolymer (polysaccharide and protein) and lipid-based micro or nanocarriers for potential applications in food and pharmaceutical industries. In particular, the function of these carrier systems in terms of encapsulation efficiency, stability, bioavailability, and bio-accessibility is critically discussed. Recently, tremendous attention has been paid to encapsulating FSVs in commercial applications. According to the chemical nature of the active compound, the vigilant selection of delivery formulation, method of encapsulation, and final application (type of food) are the key important factors to be considered in the encapsulation of FSVs to ensure a high loading capacity, stability, bioavailability, and bio-accessibility. Future studies are recommended on the effect of different vitamin types and micro and nano encapsulate sizes on bioaccessibility and biocompatibility through in vitro/in vivo studies. Moreover, the toxicity and safety evaluation of encapsulated FSVs in human health should be evaluated before commercial application in food and pharmaceuticals.
Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.
Rice bran protein concentrates (RBPC) were extracted using mild alkaline solvents (pH: 8, 9, 10). The physicochemical, thermal, functional, and structural aspects of freeze-drying (FD) and spray-drying (SD) were compared. FD and SD of RBPC had porous and grooved surfaces, with FD having non-collapsed plates and SD being spherical. Alkaline extraction increases FD's protein concentration and browning, whereas SD inhibits browning. According to amino acid profiling, RBPC-FD9's extraction optimizes and preserves amino acids. A tremendous particle size difference was prominent in FD, thermally stable at a minimal maximum of 92 °C. Increased pH extraction gives FD greater exposal surface hydrophobicity and positively relates to denaturation enthalpy. Mild pH extraction and drying significantly impacted solubility, improved emulsion properties, and foaming properties of RBPC as observed in acidic, neutral, and alkaline environments. RBPC-FD9 and RBPC-SD10 extracts exhibit outstanding foaming and emulsion activity in all pH conditions, respectively. Appropriate drying selection, RBPC-FD or SD potentially employed as foaming/emulsifier agent or meat analog.
Repressor element-1 silencing transcription factor (REST) or also known as neuron-restrictive silencing factor (NRSF), is the key initiator of epigenetic neuronal gene-expression modification. Identification of a massive number of REST-targeted genes in the brain signifies its broad involvement in maintaining the functionality of the nervous system. Additionally, REST plays a crucial role in conferring neuroprotection to the neurons against various stressors or insults during injuries. At the cellular level, nuclear localisation of REST is a key determinant for the functional transcriptional regulation of REST towards its target genes. Emerging studies reveal the implication of REST nuclear mislocalisation or dysregulation in several neurological diseases. The expression of REST varies depending on different types of neurological disorders, which has created challenges in the discovery of REST-targeted interventions. Hence, this review presents a comprehensive summary on the physiological roles of REST throughout brain development and its implications in neurodegenerative and neurodevelopmental disorders, brain tumours and cerebrovascular diseases. This review offers valuable insights to the development of potential therapeutic approaches targeting REST to improve pathologies in the brain. The important roles of REST as a key player in the nervous system development, and its implications in several neurological diseases.
Demands for high nutritional value-added food products and plant-based proteins have increased over the last decade, in line with the growth of the human population and consumer health awareness. The quality of the plant-based proteins depends on their digestibility, amino acid content, and residues of non-nutritive compounds, such as phenolic compounds, anti-nutritional compounds, antioxidants, and saponins. The presence of these non-nutritive compounds could have detrimental effects on the quality of the proteins. One of the solutions to address these shortcomings of plant-based proteins is fermentation, whereby enzymes that present naturally in microorganisms used during fermentation are responsible for the cleavage of the bonds between proteins and non-nutritive compounds. This mechanism has pronounced effects on the non-nutritive compounds, resulting in the enhancement of protein digestibility and functional properties of plant-based proteins. We assert that the types of plant-based proteins and microorganisms used during fermentation must be carefully addressed to truly enhance the quality, functional properties, and health functionalities of plant-based proteins.Supplemental data for this article is available online at here. show.
The potential of ultrasonication-driven molecular self-assembly of whey protein isolate (WPI) with chitosan (CS)/chitooligosaccharide (COS) to stabilize Pickering emulsions was examined, based on CS/COS ligands-induced partial unfolding in remodeling the Pickering particles features. Multi-spectral analysis suggested obvious changes in conformational structures of WPI due to interaction with CS/COS, with significantly higher unfolding degrees of WPI induced by COS. Non-covalent interactions were identified as the major forces for WPI-CS/COS conjugates. Ultrasonication enhanced electrostatic interaction between CS's -NH3 groups and WPI's -COO- groups which improved emulsification activity and storability of WPI-COS stabilized Pickering emulsion. This was attributed to increased surface hydrophobicity and decreased particle size compared to WPI-CS associated with differential unfolding degrees induced by different saccharide ligands. CLSM and SEM consistently observed smaller emulsion droplets in WPI-COS complexes than WPI-CS/COS particles tightly adsorbed at the oil-water interface. The electrostatic self-assembly of WPI with CS/COS greatly enhanced the encapsulation efficiency of quercetin than those stabilized by WPI alone and ultrasound further improved encapsulation efficiency. This corresponded well with the quantitative affinity parameters between quercetin and WPI-CS/COS complexes. This investigation revealed the great potential of glycan ligands-induced conformational transitions of extrinsic physical disruption in tuning Pickering particle features.